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JOHN:
Method Validation

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Accuracy, precision, repeatability, intermediate precision, specificity, limit of detection, limit
of quantitation, linearity and range
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What is specificity?
The ability of a method to measure the analyte and only the analyte in the presence of
components which may be expected to be present in the sample matrix
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What is accuracy?
Measures how close the values are to the true values
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What is repeatability?
Repeatability expresses the precision under the same operating conditions over a short
interval of time
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What is the % RSD for repeatability?
2%
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The results obtained should fall within the same range
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What is linearity?
Analyte response is directly proportional to the concentrations across the analaytical
method range
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What is range?
The concentration interval between the upper and lower levels of analyte that can be
measured with a specified precision, accuracy and linearity
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What is limit of detection?
LOD is lowest concentration of analyte that can be routinely detected: based on a signal to
noise ratio between 3 or 2:1
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What is limit of quantitation?
Lowest amount of analyte that can be quantified with suitable precision and accuracy: based
on a signal to noise ratio 10:1
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What is a system Suitability test?
Quick check to make sure that the method has been set up properly so that is it behaving as
it was when it was validated
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g a few injections to check resolution of a critical pair of peaks and an abridged
repeatability test
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STEPHEN:
Molecular spectroscopy: UV
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What are the advantages of UV spectroscopy?
Simple and inexpensive
Rapid technique
Universal
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1-10ppm)
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Explain what happens to the molecule when UV light is absorbed
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Once it reaches the
excited state, it returns to the ground state and the difference between the excited state and the
ground state is equal to the energy released
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E=hc/lambda
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H= planck constant = 6
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998 x 108

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For a Bathochromic shift (red shift)- what is the wavelength/ energy like?
Longer wavelength/lower energy
b
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c
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Does the absorbance increase/decrease for a hypochromic shift?
Decrease
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What are the 3 methods used in UV quantitation?
Calibration curve method
Comparative method
Absolute method
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What is the equation for the Comparative method?
Csample/Asample=Cstd/Astd
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Explain the absolute method?
Requires a well-established specific absorbance
Assumes linearity
Needs a high quality instrument
Accurate wavelength
Spectral resolution
Absence of stray light
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Molecular Spectroscopy: Fluorimetry

Fluorimetry
Molecular Spectroscopy: Fluorimetry
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Explain answer?
Fluorimeter is specific
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Not all drugs are florescent
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Draw what the instrumentation looks like?

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What are the disadvantages of fluorimetry?
Limited linear range
Relatively few molecules fluoresce
Prone to interferences, e
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quenching
Very solvent sensitive
Very sensitive to pH

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What is mainly used for atomic spectroscopy?
Metals
Emission is the best for alkali (Group 1): sodium, potassium, lithium
Absorption better for higher group elements (calcium, magnesium, Cn, Zn, Fe)
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The solution is sprayed into a flame
b
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Atoms are excited using thermal energy
d
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Atoms lose energy by emitting photons
f
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What are the major limitations in atomic spectroscopy?
Sample introduction system requires large volume of samples (5ml)
Not possible to analyse solid samples
High detection limits (ppm range) due to:
• Poor nebulization efficiency
• Short residence time in the flame
• Rapid dilution in the flame

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Ionisation
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Alkali and alkaline earth metals have low ionisation potential
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Spectral
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Spectral line overlap
o Line of copper at 324
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753nm
o Line of lead and sodium chloride at 217nm

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9
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Calculation question

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JOHN
Separation science in Pharmaceutical R & D – HPLC
HPLC- HIGH PERFORMANCE LIQUID CHRONATOGRAPHY
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Dependent upon :
• Purity of ingredients
• Nature of impurities
• Quantity of impurities
• Choice of ingredients
• Care with which ingredients are mixed together
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What are the 3 types of diffusion involved in separation science?
Mass transfer, eddy diffusion and longitudinal diffusion in the mobile phase
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What is the van Demeter equation?

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Use small spherical porous particles (5 microns) with a narrow particle size distribution
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What are the 3 key issues with band broadening?
Laminar flow in inter-particle channels
Knox equation to compare assess packing method
Extra column band –broadening
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What are the advantages of straight phase HPLC?
Polar analytes are more strongly retained than non-polar analytes
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Heterogeneous surface leads to peak tailing for basic analytes
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What is reversed phase HPLC?

What factors are at play in order to increase the retention time?
Increase % water or aqueous buffer
Increase alkyl chain length
Change nature of polar organic compound (e
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Effect of pH e
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Acids = decrease retention time
Bases = increase retention time
Neutrals = no change
Exam question and answer

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Separation science in pharmaceutical R&D – GC

Discuss the use of Gas Chromatography (GC) for the analysis of drugs
GC is the method of separating mixtures – suited to volatile liquids
Very high efficiency through the use of capillary GC
What is needed for GC?

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Mobile phase – Gas
Stationary phase – column packed with solid or high boiling point liquid

Originally used with ‘packed’ columns containing non-porous particles coated with a viscous,
involatile, inert liquid; typically used nitrogen (or occasionally argon) carrier gas (as mobile phase)
and hydrogen and air supplies for the flame of a flame ionisation detector
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Capillary-GC – Injections
SPLIT INJECTION - for major components
PURGE SPLITLESS INJECTION - for trace level analysis
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DIRECT INJECTION - for wide bore open tubular columns
(For all of the above, injection is into a heated inert region provided by a glass liner which fits in the
injection head)
ON-COLUMN (cool) INJECTION - for high boiling or thermally labile material

PACKED GC INJECTION PORT - adapted to accommodate wide bore capillary column
Septum

Glass liner

Wide bore fused silica capillary column
1/4” nut
Reducing ferrule 1/4” x 1/16”

SPLIT / SPLITLESS INJECTION - For splitless injection, the valve is closed and a very volatile solvent is
used; latent heat of evaporation of the solvent cools down the head of the column so that the trace
analyte is ‘focused’ before warming up again and passing down the column
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As a substance leaves the column, it burns in this flame producing ions which can be detected
by measuring the electro conductivity of the flame
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An alkali metal salt is (caesium bromide or rubidium sulphate) impregnated on a wire grid, on porous
ceramic or compressed into a tip in close proximity to the hydrogen flame
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Pros:
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selective towards N and P containing compounds
can be operated in a N,P mode or a P mode
has low limits of detection for N and P (sub-pg)

Cons:
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has limited long term stability

Electron-Capture detector: The internal chamber of the detector is lined with a b-emitter (3H or 63Ni)
in a tin foil
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g
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The resultant ionisation current is reduced in the presence of an analyte capable of capturing
electrons to form ions
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The electron capture detector is most sensitive for halogen-containing analytes with
limits of detection in the range, 10-12 - 10-13 g
It has a limited linear dynamic range (x50 - x100)
Attention to detail is required e
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carrier gas quality, cleanliness, maintenance of
constant temperature and sample clean-up procedures

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Pros:
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The ECD is highly selective toward compounds with electronegative functional groups
– Halogens
– Nitro groups

Cons:
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Relatively insensitive to amines, alcohols, and hydrocarbons
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Over all GC
Pros:
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Universal (have to make substance volatile)
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High efficiency
Wide range of sensitive detectors available
Can use long columns due to low resistance of flow
Thin film gives rapid mass transfer

Cons:
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Not specific
Limited – can’t analyse substances with chromophores
Small internal volume means limited sample loading

Separation science in pharmaceutical R&D- TLC

(a) Silica layer
(b) glass plate
(c) development tank
(d) eluting solvent
(e) Plate after development with spots resolved

The Rf value is used to define the position of an analyte after it has migrated along the TLC plate
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If the spot has an Rf value of 1, this means that has not been retained
on the TLC stationary phase, and the mobile phase would have to be adjusted to allow the analyte to
be retained on the stationary phase
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A non-destructive method is to place the TLC plate under a UV lamp
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There are also a number of reagents to help visualise the components without UV light
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The developing reagent used can either be a general purpose reagent or a reagent which
depends on the chemistry of the components in the sample
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A few crystals of
iodine are placed in the bottom of a glass jar and the TLC plate is placed in it
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Another general purpose reagent is a molybdate spray which is sprayed onto the TLC plate
and then heated at 105C for 5 minutes
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An example of a more specific way of visualising components is by using Gibb's reagent
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Key features of TLC
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multiple samples may be run in parallel
all sample constituents may be visualised
cheap and easy to run
limit tests
identity tests for a
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g
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What are the applications of ion exchange?
Inorganic ions, strong organic bases, drugs in biological fluids, proteins
What are the features of ion pair?
Pairing ions used are e
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long chain alkyl sulphonic acids, tetrabutyl ammonium hydroxide,
selectivity may be controlled by pH, retention controlled by % organic, (ion-pair), carried out on
alkyl-silicas
What are advantages and disadvantages of ion pair?
Advantages

Disadvantages

Large number of variable

Long equilibrium times

Very selective

Columns not suitable for RPLC re-use

Retention varies with (ion pair) in a
Predictable fashion

What are applications of ion pair?
Applications: Hydrophilic ionic compounds, class separations, simultaneous determination
of acids, bases and neutrals (indirect UV visualisation)
What are the features of ligand exchange?
Feature: 7
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5, normally Cu2+ , large number of variables, v
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What are the applications of ligand exchange?
Applications: Amino acids, (amino alcohols), α-hydroxyl acids, chiral separations

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For protein separation what are the suitable models of LC?
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size exclusion

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ion exchange

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reversed phase (hydrophobic interaction LC)

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affinity

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(perfusion (wide macropore channels and still micropores))

What are the conditions needed to test hydrophobic interactions?
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Polar Stationary Phase

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Aqueous buffer mobile Phase

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Use Gradient Elution

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(high ionic strength to low ionic strength)

What are the conditions needed for ion-exchange LC for proteins?
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Polar Stationary Phase

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Aqueous buffer mobile Phase

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Use Gradient Elution

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(low ionic strength to high ionic strength; stronger elution in the presence of competing
ions)

How to select
correct method?

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API ANALYSIS
What does API stand for?
Active pharmaceutical ingredient
What are the different methods of analysis of API?
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Appearance test: colour (can see impurities), crystallinity
Identity test, IR
Assey, RP-HPLC
Related substances, RP-HPLC
Trace metals, atomic spectroscopy (AA, flame photometry (ICPMS)
Trace inorganics, sulphated ash test
Water, karl fisher titration
Residual solvents, headspace GC

How does Karl fisher titration work?
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The titrations are based on iodine oxidising sulfur dioxide in the presence of water
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The end point is found by the
detection of excess iodine which occurs when there is no longer any water present
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Selectivity for water
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Easy sample preparation
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suitable for analysing:
- Solids, Liquids, Gases

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Suitability for automation
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Cool in desiccator and weigh

accurately
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Moisten with
sulphuric acid
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Leave to cool
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Transfer to electric furnace
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Calculate percentage
of residue
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g
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What is derivatisation?
Derivatisation is the process of chemically modifying a compound to produce a new
compound which has properties that are suitable for analysis using a GC
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Masking these groups dramatically increases volatility
Very volatile compounds can be made less volatile
Derivatisation can introduce groups to provide specific increases in detection levels
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What are the types of derivatisation?
Silylation – readily volatises the sample and is the most prevalent method
Alkylation- used as the first step to further derivatisations or as a method of protection of
certain active hydrogens
Acylation – commonly used to add fluorinated groups (ECD)
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The better the leaving group the better
the silylation
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• Sample and solvents must be dry
• Vacuum filtration

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Solvents should be as pure as possible – where pyridine is the most commonly used
solvent
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What are the silylation reagents?
BSA N,O-Bis(trimethylsilyl)acetamide - forms highly stable TMS derivatives with most organic
functional groups under mild reaction conditions
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TMSI- trimethylsilylmidazole
Potent – reacts with alcohols and phenols
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What are the three steps involved in mass spectroscopy?
Ionisation- Initial sample might be a solid, liquid or a gas
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Heating up a metal coil until it releases electrons
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Acceleration- All species have the same kinetic energy (like starting a race with all the runners on
the same line)
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Deflection- ion beam passes through a magnetic field
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Detection- Data is plotted of different masses
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What does m/z stand for?
Mass to charge ratio
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What are the pros of Mass spec?
Sensitive
Good for identifying unknown components in a sample

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